Current efforts are directed towards understanding the relationship between cell cycle regulation and differentiation using the Drosophila compound eye as a model system. Analysis of roughex (rux), a mutation previously shown to be necessary for G1 arrest during eye development, demonstrated that rux functions by regulating the accumulation of the G2 cyclin, Cyclin A (CycA), by a post-transcriptional mechanism, most likely at the level of protein stability. Overexpression of Rux protein blocks mitosis and leads to mis-localization of CycA and CycB to the nucleus, where they are subsequently degraded. Recent experiments show that Rux can bind directly to CycA both in a mammalian two-hybrid system and in co-immunoprecipitation experiments from Drosophila Schneider cells expressing both proteins. Co-expression of the proteins in Schneider cells also leads to mislocalization of CycA to the nucleus, although in this system CycA remains stable. Deletion analysis of Rux has defined two regions important for CycA binding; both of these regions contain a motif, RXL, which has been shown in mammalian cyclin kinase inhibitors to be necessary for binding to cyclins. Mutation of the first RXL motif in the N-terminal region of the protein completely abolishes binding to CycA in the mammalian two-hybrid assay, while mutation of the second RXL motif reduces binding in the same assay. A bipartite nuclear localization signal (NLS) was also identified; when this domain is deleted, CycA remains cytoplasmic in Schneider cells, although binding of Rux to CycA is still detected in the two-hybrid assay. In the developing eye, deletion of the Rux NLS also resulted in cytoplasmic accumulation of CycA; in addition, CycA protein was not destabilized. Cytoplasmic CycA was not competent for mitosis and S phase was prolonged in cells expressing the Rux NLS mutant, suggesting that CycA must translocate to the nucleus for its S/G2 functions. These findings are currently submitted for publication.We are also initiating a study of the interaction between Rux and the G1 regulator Cyclin E (CycE). We previously showed a physical interaction between Rux and CycE in vitro and in a yeast two- hybrid system. In addition, overexpression of CycE in flies results in down-regulation of Rux protein and the re-appearance of active CycA, suggesting that the role of CycE is to down-regulate Rux as cells enter S phase to release CycA for its S/G2 function. Rux contains four consensus sites for phosphorylation by cyclin dependent kinases (Cdk), and Rux is a good target for phosphorylation by CycE/Cdk2 complexes immunprecipitated from Drosophila embryos. Mutation all four consensus Cdk phosphorylation sites abolished the ability of CycE to down- regulate Rux in vivo, suggesting that CycE netatively regulates on Rux via these sites.A third project in the lab is the cloning and molecular and phenotypic characterization of one genetic suppressor of the rux mutation, S42/S56. A P element allele, l(2)00632, was identified and used to isolate cDNAs surrounding the region of the insertion. A 9-10 kb cDNA was isolated using 5 and 3 RACE (Rapid Amplification of cDNA Ends). Preliminary phenotypic analysis shows that the S42/S56 mutant strongly resembles mutations in cycA,suggesting that S42/S56 functions in a pathway to regulate positively Cyclin A activity during development.Future plans include the further molecular and phenotypic characterization of S42/S56, and the cloning and analysis of other identified suppressors of rux. The further biochemical characterization of Rux is also planned, including further analysis of the role of the consensus Cdk phosphorylation sites on the stability and/or activity of the Rux protein. - cell cycle, cyclin, cyclin dependent kinase, cyclin kinase inhibitor, Drosophila, eye development, mitosis, G1, - Neither Human Subjects nor Human Tissues